| Objective:Glioma is the most common intracranial primary tumor with high proliferation rate,strong invasiveness,low apoptosis rate,poor prognosis and easy recurrence.β-sitosterol（BS）is a natural phytosteroid widely found in plants,which has been effective in cancer prevention and treatment,cardiovascular protection,liver protection,anti-inflammatory and antioxidant,antiviral,and diabetes treatment.This study aims to explore the inhibitory effect of BS on glioma,further analyze its target,and provide new ideas and insights for the treatment of glioma.Method:In vitro experiments:Human astroblastoma U87 cell line was selected,and MTT was used to detect changes in U87 cell activity after BS intervention with different concentration gradients and time gradients,so as to further clarify the optimal concentration and time of intervention for U87 cells.The effects of different concentrations of BS on colony formation of U87 cells were determined by plate cloning.Hoechst33342 staining and Annexin V-FITC/PI method were used to investigate the effects of different concentration gradients of BS on apoptosis of U87 cells.The cell cycle changes of U87 cells after BS intervention were measured by flow cytometry.The effect of BS on the migration behavior of U87 cells could be proved by the wound healing method and Transwell TM method.In addition,the expression of apoptosis and migration-related proteins in U87 cells after BS treatment was mutually verified with the above experiment,and western blot was used to determine the expression.The target and signal pathway of BS on U87 cells were screened by using the method of network pharmacology and transcriptomics,and the above results were verified by molecular docking and Western-blot.In vivo experiment:The axillary glioma model of C57 BL/6 mice was constructed and divided into the Control group and the BS group.The time of tumor formation,average tumor weight,tumor size,important organ index,and animal survival status were observed.HE staining was used to observe the histomorphological changes before and after BS treatment,and to evaluate the pathological changes in tumor tissues after BS intervention.Result:In vitro experiments:（1）Cell morphology results showed that compared with the control group,the number of U87 cells in the BS group was significantly reduced,and the irregular cell contour,cell contraction,and even shedding were significantly dependent on drug administration time and concentration;（2）After BS treatment for 24 h,48 h and 72 h,the IC50 of U87 cells was 35.82μM,22.75μM,and 9.43μM,respectively.（3）Hoechst 33342 staining and Annexin V-FITC/PI assay indicated that BS promoted apoptosis of U87 cells in a certain dose range（P<0.001）;（4）Flow cytometry showed that BS inhibited U87 cells in G2/M phase in a dose-dependent manner（P<0.001）;（5）Plate cloning experiment showed that BS inhibited the proliferation and colony formation of U87 cells in a dose-dependent manner（P<0.001）;（6）Scratch and Transwell TM results showed that BS inhibited U87 cell migration in a dose-dependent manner（P<0.001）;（7）Western-blot results showed that apoptosis protein bax/bcl-2 ratio and cleaved caspase-3 expression level increased,EMT process-related protein E-cadherin expression increased,β-catenin and Vimentin expression decreased,in a dose-dependent manner.The results were consistent with other phenotypic experiments（P<0.001）;（8）Combined with network pharmacology and transcriptology results,EGFR/MAPK signaling pathway may be involved in the regulation of BS on glioma;（9）Molecular docking experiments confirmed thatβ-sitosterol had good binding ability to target protein;（10）Western-blot evidence of signal pathway screening results:BS can effectively down-regulate the expression of key target proteins of EGFR/MAPK signaling pathway:EGFR,SOS1,and P-ERK1/2（P<0.01）.In vivo experiment:（1）We conducted a holistic observation of the two groups of animal models,and found that compared with the no-drug-intervention control group,the BS group had a good mental state,and the average tumor weight and tumor volume decreased,with significant statistical differences（P<0.001）,and there was no significant difference in body weight and important organ index between the two groups;（2）HE staining results showed that the Control group had disordered tissue structure and more nuclear mitosis,while the BS group had orderly tissue structure and uniform cell distribution.Conclusion:β-sitosterol can inhibit the proliferation and migration of U87 glioma cells in vitro by down-regulating the EGFR/MAPK signaling pathway,induce the apoptosis and cell cycle arrest of U87 cells,and significantly inhibit the growth of mouse transplanted glioma.These results suggest thatβ-sitosterol can play a therapeutic role in glioma by down-regulating EGFR/MAPK signaling pathway. |
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